! Project Summary A major clinical issue in most cancers is relapse. Patients often respond very well to chemotherapy, and can go years without any sign of disease, but a subset of patients will invariably re- develop their cancer with a poor final prognosis. Relapse occurs because our current chemotherapies are unable to reliably and completely eliminate tumor propagating cells, also known as cancer stem cells. These cells are unique among the tumor cell population in that they can self-renew, meaning that they can replenish a tumor cell population indefinitely, similar to the role of the normal tissue stem cells in tissue and organ maintenance. Preventing self-renewal in tumor propagating cells would cause them to terminally differentiate, thereby blocking their ability to form relapse. Unfortunately, self-renewal of tumor propagating cells is not well understood, precluding rational drug design. A major issue in regards to studying these cells is their rarity; they often comprise 1 in every 105-107 cells within the total tumor cell population in human cancers and mouse models, and culture ex vivo alters their self-renewal capability. Researchers necessarily rely on FACS enrichment based on certain cell surface markers, but this biases towards the cells expressing the markers and excludes some subsets of self-renewing cells. The goal of this project is to have a major impact in the biomedical field by defining self-renewal in tumor propagating cells in a completely unbiased manner. We will use leukemia propagating cells as a model and determine how these cells differ from normal hematopoeitic stem cells, which can also self- renew, and how are they unique from other leukemic cells that cannot self-renew. Based on these data, we will find ways to detect leukemia propagating cells in patients, and identify drugs that can inhibit their self-renewal ability. Initially, we will use a panel of high self-renewing acute lymphoblastic leukemias and normal hematopoietic stem cells isolated from zebrafish models, which will allow us to use single cell RNA sequencing to identify the unique gene expression profile of self-renewing leukemia propagating cells without the need for FACS enrichment. We will translate our findings to human cells to build a biomarker panel that can detect the frequency of self-renewing cells in patient samples, and tell us whether chemotherapy has successfully eliminated them. We will also characterize the stem cell niche in leukemias with high and low self-renewal rates, to identify how the niche is regulating self-renewal rate. Finally, we will use transplantation approaches in zebrafish, as well as new zebrafish models in which the leukemia propagating cells are fluorescently labeled, for high-throughput, in vivo drug screens to identify compounds that impair self-renewal. In total, this project will provide an unbiased genomic and functional analysis of tumor propagating cells, allowing us to answer fundamental questions about the biology of this important tumor cell type.